Our overall goal is to identify and characterize new molecular mechanisms of integrin biological function and intracellular signaling. Integrin receptors and the extracellular matrix proteins to which they bind play crucial roles in a wide variety of cellular interactions that are important in human development, health, and disease. Integrins mediate or regulate cell adhesion, migration, and extracellular matrix assembly, as well as cell surface control of the cytoskeleton, gene expression, and growth. [unreadable] We are addressing the following general questions focused on integrin-matrix interactions:[unreadable] 1. How do integrins, the extracellular matrix, and the cytoskeleton interact to produce cell migration?[unreadable] 2. What are the integrin signaling pathways involved in biological functions such as migration? [unreadable] 3. How do cells assemble a fibronectin-based extracellular matrix?[unreadable] 4. What are the signaling and biological differences between traditional two-dimensional (2D) cell cultures and various 3D matrices characteristic of microenvironments in vivo?[unreadable] We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, fluorescence time-lapse microscopy, and mutational analysis. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins, including paxillin, vinculin, talin, and tensin, in an ongoing program to analyze their functions in integrin-mediated processes. We have been focusing particularly on functions of integrins and associated molecules in cell migration and invasion, matrix assembly, and matrix remodeling. [unreadable] In the previous fiscal year, we had established that the precise level of active Rac is a critical general regulator of the directionality of cell migration. Cells with 30% or greater reductions in Rac activity were shown to migrate in straight paths with a high degree of persistence in one direction in a variety of cell types. We subsequently extended these studies to establish detailed protocols and extensive sets of controls for the experimental manipulation of the activity of Rho GTPase family members by RNA interference using siRNA. We found that individual GTPase knockdown by siRNA produces a surprisingly proportional down-regulation of activity of just that Rho family GTPase. Our approach currently provides a specific method for dissecting the role of individual members of this important signal transduction family.[unreadable] The most-studied mediator of integrin cytoskeletal and signaling interactions is the scaffold protein termed focal adhesion kinase (FAK). We had previously published a series of studies on FAK concerning its roles in adhesion and migration in normal and tumor cells. We completed collaborations with groups studying other roles of FAK. We found that FAK functioned cooperatively with the recently described protein JSAP1, which serves as a signaling scaffold for the MAP kinase JNK. This intermolecular cooperation between major signaling pathway components stimulates cell migration. Yet another pathway implicated in cell migration by normal and malignant cells is the MMP (matrix metalloproteinase) system. FAK was found in another collaboration to regulate the release of MMP-2 and MMP-9 by human T cells. A novel finding was that different regions of FAK have distinct regulatory functions in protease regulation, identifying new complexity in its downstream regulatory functions.[unreadable] We initiated a relatively high-risk project 4 years ago to develop cutting-edge imaging methods to visualize actin and integrin dynamics at the extreme leading edge of migrating cells, particularly in lamellipodia and filopodia -- the thin extensions of the lamella of migrating cells. Working with the Carl Zeiss microscope company and in collaboration with James Galbraith at NINDS, new imaging and quantification algorithms have been developed. The goal was to determine how migrating cells such as fibroblasts and neurons regulate local cell protrusions to produce migration and how they probe their local microenvironment to establish the very first attachments to the substrate. The goal was achieved using a combination of high-resolution confocal visualization with quantification of actin and integrin dynamics using optical and quantitative image analyses including local photoactivation of actin fluorescence. Local sites of actin polymerization shuttle rapidly transversely along the leading edge of migrating cells to make "sticky fingers," i.e. local membrane protrusions with integrins clustered into tiny precursors of adhesions that are locally activated to bind fibronectin. These tiny cellular "sticky fingers" probe back and forth to establish the very first cell adhesions in fibroblasts and neurons. These findings establish the concept that actin dynamics regulate the sites of forward cell advance and subsequent attachment via integrins during pathfinding by migrating cells.[unreadable] We completed a study identifying markers for the tiny cellular structures termed invadopodia that serve as foci of tumor cell proteolysis. Our study characterized a novel molecular time sequence for formation of invasion-mediating organelles termed invadopodia. To invade, human carcinoma cells first clustered actin and cortactin, which led to clustering of MT1-MMP, a protease reportedly necessary for tumor cell invasion. Immediately thereafter, associated proteolytic holes appeared in the substrate, followed by departure of the organizing cortactin and actin, leaving behind functional MT1-MMP clusters that continued local proteolysis. Transfection and gene silencing analyses indicated that cortactin is needed to initiate the process and that MT1-MMP is needed later for proteolysis (but not for initial invadopodial organization). This study provided the first live-cell time-course of the dynamics of molecules in invadopodia of human breast and other cancer cells, and it suggests that targeting either the cytoskeletal component or the proteolytic clustering component of the process of invadopodial function could inhibit invasion. [unreadable] These ongoing studies on the functions of integrins and associated molecules in development and cancer invasion center upon our ability to image live-cell molecular dynamics of the crucial first protrusions, contacts, and sites of local matrix degradation. All of these processes will need to be analyzed in parallel in real time to be able to understand the mechanisms of in vivo cell migration and invasion. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.[unreadable] In summary, our Section is elucidating mechanisms by which integrins function in cell adhesion, migration, invasion, and matrix assembly. A unifying theme has been the importance of understanding the dynamics of local cell-matrix interactions. Such analyses are important for understanding how adhesion receptors control cell and tissue movements, organization, growth, and development. We will continue to search for novel mechanisms and modulators. Knowledge of these basic processes should facilitate creative approaches to therapy, particularly in the fields of tissue engineering and cancer biology.